In the early part of the 20th century, vitamin B3 was identified as a component missing from the diet of pellagra patients. Supplementation with nicotinic acid, or niacin, ameliorated the symptoms of pellagra, and prevented the onset of this condition in areas where it was prevalent. The biochemical role of niacin was elucidated in the 1930s, when it was found to be critical for the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a compound essential for cellular respiration (Preiss, J.; Handler, P. Biosynthesis of Diphosphopyridine Nucleotide I. Identification of Intermediates J. Biol. Chem. 1958 233, 488-492.; Preiss, J.; Handler, P. Biosynthesis of Diphosphopyridine Nucleotide II. Enzymatic Aspects J. Biol. Chem. 1958 233, 493-500). The precise role of NAD in cellular respiration is well understood. As glucose and fatty acids are oxidized, NAD can accept a hydride equivalent, which results in its reduction to NADH. NADH can donate a hydride equivalent, resulting in oxidation back to NAD. These reduction-oxidation cycles use NAD for the temporary storage of hydride ion, but they do not consume NAD. There are other enzymes that use NAD in a different manner, and for purposes not directly related to energy production. Poly-ADPribose polymerases (PARPs), ADPribose transferases (ARTs), and sirtuins all catalyze reactions that release nicotinamide from NAD. This reaction generates a significant amount of energy, similar to ATP hydrolysis. The reverse reaction does not occur readily, so NAD must be replenished by other mechanisms (Bogan, K. L.; Brenner, C. Nicotinic Acid, Nicotinamide, and Nicotinamide Riboside: A Molecular Evaluation of NAD+ Precursor Vitamins in Human Nutrition Annu. Rev. Nutr. 2008, 28, 115-130).
Niacin (or nicotinic acid (pyridine-3-carboxylic acid)), and its amide niacinamide (or nicotinamide (pyridine-3-carboxamide)) are converted to NAD in vivo. Nicotinamide adenine dinucleotide (NAD+) is a key coenzyme found in all living cells that functions as an electron carrier in oxidative and reductive biochemical reactions occurring throughout metabolism. In mammals, niacinamide, rather than niacin, may be the major NAD precursor. The set of biosynthetic transformations from niacinamide to NAD is shown in FIG. 1. The rate limiting step for this pathway is the formation of the bond between niacinamide and 5-phosphoribose-1-pyrophosphate (PRPP), and it is catalyzed by nicotinamide phosphoribosyl transferase (NAMPT) (Revollo, J. R.; Grimm, A. A.; Imai, S.-I. J. Biol. Chem. 2004, 279, 50754-50763). The NAMPT pathway is thought to be the most efficient route known for nicotinamide recycling. Niacin enters into a similar set of transformations, but in a final step, the carboxylic acid must be converted to a carboxamide to produce NAD. The biosynthesis of NAD from niacin follows the Preiss-Handler pathway (FIG. 1).
In 1982, nicotinamide riboside (NR) was investigated as a NAD precursor in prokaryotes (Liu, G.; Foster, J.; Manlapaz-Ramos, R.; Loivera, B. M. “Nucleoside Salvage Pathway for NAD Biosynthesis in Salmonella typhimurium” J. Bacteriol. 1982, 152, 1111-1116). In contrast to niacin, exogenously supplied NR is hypothesized to bypass the first and most energy-consuming part of both the Preiss-Handler pathway and the NAMPT pathway (FIG. 1). Although NR appears to be a natural precursor for NAD, it likely represents only a small amount, if any, of NAD biosynthesis owing to the apparent scarcity of NR in dietary sources. NR contains a high energy glycosidic bond that is spontaneously labile in aqueous solution, yielding nicotinamide and ribose decomposition products. This spontaneous reaction occurs over the course of hours or days depending on the exact ambient conditions, but it makes any naturally occurring NR difficult to keep in food sources, while nicotinic acid or nicotinamide are considerably more stable and easy to prepare and administer. NR has been reported to occur in milk (Bieganowski and Brenner (2004) Cell 117: 495-502) and beer, but the amounts typically present are probably too small to be nutritionally significant.
Currently, NR supplementation is limited by the available commercial supply. NR supplementation could represent a dietary alternative to niacin, with the advantage of being a more efficient NAD precursor. By taking advantage of a natural pathway to synthesize NAD while consuming less energy, NR could offer benefits for human health. Cells are constantly subject to damage by normal environmental factors, and they have evolved repair mechanisms to continuously reverse this damage. The repair mechanisms consume NAD by scission of the high energy glycosidic linkage to produce species such as poly-ADPribose and ADP-ribosylated proteins. In severely damaged cells, energy stores are not sufficient to produce the NAD necessary to maintain homeostasis, and the damage becomes irreversible. Therefore, an energy-rich NAD precursor such as NR may be able to address cell and tissue damage at the molecular level.
NR can be difficult to isolate from natural sources, so it is typically produced by chemical synthesis. The first chemical synthesis was accomplished by Todd and co-workers in 1957 (Haynes, L. J.; Hughes, N. A.; Kenner, G. W.; Todd, A. J. Chem. Soc. 1957, 3727-3732). This group produced NR chloride as a mixture of α and β anomers about the glycosidic linkage in an approximately 1:4 ratio. The product was described as a hygroscopic oil that could not be crystallized. Other investigators who isolated NR chloride from biochemical sources also described it as a hygroscopic oil (Schlenk, F. “Nicotinamide Nucleoside” Naturwiss. 1940, 28, 46-47; Gingrich, W.; Schlenk, F. “Codehydrogenase I and Other Pyridinium Compounds as V-Factor for Hemophilus Influenzae and H. Parainfluenzae” J. Bacteriol. 1944, 47, 535-550). Significantly, biochemical syntheses should have produced only the natural β-anomer, though the exact stereochemical arrangement was not determined. Later reports confirmed the hygroscopic, amorphous nature of NR chloride (Jarman, M.; Ross, W. C. J. J. Chem. Soc. C, 1969, 199-203; and Atkinson, M. R.; Morton, R. K.; Naylor, R. Synthesis of Glycosylpyridinium Compounds from Glycosylamines and from Glycosyl Halides J. Chem Soc. 1965, 610-615). Other groups investigated alternative NR anions. One synthesis described the anomerically pure NR bromide salt as crystalline, but the product was not adequately described to ascertain whether the material was truly crystalline or merely an amorphous solid (Lee, J.; Churchill, H.; Choi, W.-B.; Lynch, J. E.; Roberts, F. E.; Volante, R. P.; Reider, P. J. “A chemical synthesis of nicotinamide adenine dinucleotide (NAD+)” Chem. Commun. 1999, 729-730). Subsequently, other NR salts were prepared and solids were obtained, though they were never described as crystalline (Tanimori, S.; Ohta, T.; Kirihata, M. An Efficient Chemical Synthesis of Nicotinamide Riboside (NAR) and Analogues Bioorg. Med. Chem. Lett. 2002, 12, 1135-1137; Franchetti, P.; Pasqualini, M.; Petrelli, R.; Ricciutelli, M.; Vita, P.; Cappellacci, L. Bioorg. Med. Chem. Lett. 2004, 14, 4655-4658; Yang, T.; Chan, N. Y.-K.; Sauve, A. A. J. Med. Chem. 2007, 50, 6458-6461). In addition, methods of preparing nicotinamide riboside from enriched natural sources, such as a genetically engineered yeast strain, have been described (see, WO 2010/111111).
While nicotinamide riboside itself is useful as an efficient precursor of NAD+ to elevate NAD+ levels and improve cell and organismal health, its bioavailability under various modes of administration may be limited. Accordingly, nicotinamide riboside analogs with improved bioavailability and optimal tissue selectivity are desirable, however such compounds may also prove toxic to cells. For example, benzamide riboside is a well-known antitumor agent that is metabolized to the active NAD analogue benzamide adenine dinucleotide, which inhibits certain NAD-dependent dehydrogenases, such as malate dehydrogenase and glutamic acid dehydogenase, which may cause adverse effects. Accordingly, there is a need for NAD+ elevating agents that are bioavailable, stable, effective at NAD+ elevation in the desired tissue(s) and safe from adverse effects on NAD+-dependent biological processes.